It has been proposed that dysregulation of the fuel sensing enzyme AMPK could be both a pathogenic factor for type 2 diabetes and other disorders associated with the metabolic syndrome and a target for their therapy. Nutrient excess reduces AMPK activity and causes insulin resistance (IR) in incubated rat EDL muscle (high glucose or leucine), cultured HepG2 cells (high glucose) and muscle and liver of rats infused with glucose (high glucose and insulin). In all three models, we have found that these changes are associated with a decrease in SIRT1 and activation of mTORC1, two other fuel sensing molecules that have been linked to the metabolic syndrome. Where examined, decreased AMPK activity appeared to be the initial event and activation of AMPK prevented all of these changes from occurring, including the IR. In separate studies, others have shown that AMPK can activate SIRT1 and we have found that SIRT1 can activate AMPK, suggesting the existence of a SIRT1/AMPK cycle in which these molecules jointly govern many key enzymes and transcriptional regulators. Upon this background, studies will be performed with the following specific aims: (1) To examine at a mechanistic level how AMPK, SIRT1 and mTORC1 functionally interact with each other in incubated muscle and cultured HepG2 cells. In doing so, we will also test the hypothesis, suggested by our preliminary data, that the initial effect of a nutrient excess is to downregulate AMPK by causing it to interact with a protein phosphatase. (2) To determine in the same models how dysregulation of the three molecules leads to IR and under what conditions the IR is associated with inflammation, oxidative and ER stress and mitochondrial abnormalities. In the process, we will evaluate AMPK, SIRT1 and mTORC as targets both for preventing and treating IR using metabolic, pharmacological and genetic approaches and (3) To test whether the mechanisms defined in vitro are operative in muscle, liver and adipose tissue in vivo using the glucose- infused rat and genetically modified mice as models. Collectively, these studies will provide novel information about fundamental mechanisms by which nutrient excess leads to IR in mammalian tissues. They are especially timely since therapies that activate AMPK and SIRT1 are presently being used or are undergoing trials for disease prevention and treatment in humans.